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Objectives The risk factors and outcomes associated with persistent bacteraemia in Gram-negative bloodstream infection (GN-BSI) are not well described. We conducted a follow-on analysis of a retrospective population-wide cohort to characterize persistent bacteraemia in patients with GN-BSI. Methods We included all hospitalized patients >18 years old with GN-BSI between April 2017 and December 2021 in Ontario who received follow-up blood culture (FUBC) 2–5 days after the index positive blood culture. Persistent bacteraemia was defined as having a positive FUBC with the same Gram-negative organism as the index blood culture. We identified variables independently associated with persistent bacteraemia in a multivariable logistic regression model. We evaluated whether persistent bacteraemia was associated with increased odds of 30- and 90-day all-cause mortality using multivariable logistic regression models adjusted for potential confounders. Results In this study, 8807 patients were included; 600 (6.8%) had persistent bacteraemia. Having a permanent catheter, antimicrobial resistance, nosocomial infection, ICU admission, respiratory or skin and soft tissue source of infection, and infection by a non-fermenter or non-Enterobacterales/anaerobic organism were associated with increased odds of having persistent bacteraemia. The 30-day mortality was 17.2% versus 9.6% in those with and without persistent bacteraemia (aOR 1.65, 95% CI 1.29–2.11), while 90-day mortality was 25.5% versus 16.9%, respectively (aOR 1.53, 95% CI 1.24–1.89). Prevalence and odds of developing persistent bacteraemia varied widely depending on causative organism. Conclusions Persistent bacteraemia is uncommon in GN-BSI but is associated with poorer outcomes. A validated risk stratification tool may be useful to identify patients with persistent bacteraemia. [ABSTRACT FROM AUTHOR]